PROJECT SUMMARY This proposal aims to develop live-cell, multi-channel imaging tools that can visualize?continuously, and in real time?nanoparticle interactions with cellular components. We will focus on several different time windows, where early time periods will monitor nanoparticle-cell membrane binding and uptake and later times will track endosomal accumulation and escape. The ability to resolve temporally and spatially how particle size/shape and ligand density affects interactions in 3D is critical for determining structure-activity-relationships and mechanism of action in live cells. To characterize interactions of functional nanoparticles with different cellular structures at physiologically relevant times, we propose to design a multi-channel optical microscope integrated with an opto- splitter and custom live-cell imaging chamber. Simultaneous images can be acquired in different channels of the fluorescence of dye-labeled organelles and dye-labeled ligands on the particles as well as differential interference contrast (DIC) signals of whole cells, cellular components, and nanoparticle cores. Correlation of structural and functional images provides a powerful window into how local nanoconstruct interactions can mediate a biological response. For model systems, we will compare gold nanoconstructs with oligonucleotide ligand shells of both targeting (DNA aptamers) or non-targeting (siRNA) properties. Nanoparticle shape enables a unique handle to probe rotation and orientation of intracellular particle interactions. This work can bridge a gap in understanding the behavior of nanoconstructs intracellularly and how the integrity and presentation of oligonucleotides in ligand shells affects targeting and other processes such as endosomal escape, which is critical to assess therapeutic efficacy.